Tween 20

Low-gluten wheat flour and sugar were purchased from Angel Yeast Co., Ltd (Yichang, China). Milk was obtained from Weidendorf (Shanghai, China). Eggs were bought from a local supermarket. Sodium bicarbonate food grade, ammonium bicarboante (food grade), Tween-20, sodium dodecyl sulfate (SDS), β-mercaptoethanol, tris (hydroxymethyl) aminomethane-HCl (Tris–HCl), hydrogen peroxide (H₂O₂), folin-phenol,2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium persulfate (K₂S₂O₈), alicylic acid, ferrous sulfate and 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic) acid (ABTS) were obtained from Sinopharm Chemical Reagent Factory (Shanghai, China). Sodium acetate, 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride and ethanol were purchased from Sigma-Aldrich (St Louis, MO). Standard N-ε-carboxymethyl lysine (CML) was purchased from Toronto Research Chemicals (Toronto, Canada). In addition, solvents and chemicals such as methanol (HPLC grade), chloroform (HPLC grade) and sodium borohydride solution (pH 13–14) were supplied by Fisher Scientific (Fairlawn, NJ, USA). The other chemicals were of analytical grade.

Cells were washed twice with PBS and incubated with 2× loading buffer (0.1M Tris-HCl,4% (w/v) SDS,0.2M DTT,3mM Bromophenol blue,20% Glycerol) to lyse the cells and extract the total cellular protein. The extracted total protein was heated at 95°C for 10 min and sonicated for 10 min, after which the protein sample was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Pall Corporation, USA). The membranes were blocked with PBS containing 5% nonfat dry milk and 0.05% Tween 20 (Sinopharm Chemical Reagent Co.Ltd., China) for 2 h at room temperature. Next, they were incubated with the primary antibody (1403113, PEDV-N Monoclonal Antibody, medgene, USA; ET1601-4, Anti-GAPDH, HUABIO, China; ER1802-12 Anti-LSD1, HUABIO, China) at room temperature for 2 h. After washing the membranes with PBS containing 0.05% Tween 20 (0.05% PBS-T), they were incubated with the corresponding secondary antibody (GB23303, HRP-labeled goat anti-rabbit IgG; GB23301, HRP-labeled goat anti-mouse, Servicebio, China) at room temperature for 1 h. Finally, the membranes were detected using a chemiluminescent imaging system (BIO-RAD, USA).

Bovine serum albumin (BSA), casein, Tween 20, and other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd., and TMB (3, 3′, 5, 5′ tetramethylbenzidine) substrate (containing H₂O₂) was purchased from Neogen (Lexington, KY, USA). PSA, anti-PSA antibody (PSA mAb), and anti–PSA antibody peroxidase conjugate (PSA mAb-HRP) were purchased from Fitzgerald Industries International (Acton, MA, USA). Hydrogen tetrachloroaurate (III) hydrate (HAuCl₄) was purchased from J&K Scientific Ltd. Normal human sera were obtained from Renji Hospital, School of Medicine, and Shanghai JiaoTong University. The 16–channel screen–printed carbon electrode (16-SPCE) was purchased from Zhejiang Nanosmart Biotechnical Co., Ltd. (Ningbo, China).
The buffer solutions involved in this study are as follows: the antigen was dissolved in 0.01 M phosphate-buffered saline solution (PBS). The coating antibody was dissolved in a 0.01 M sodium carbonate (CB) buffer; the SPCE was pretreated in 0.1M phosphate buffer (PB) solution, and the PBST solution was used to clean the modified SPCE. All chemical reagents were prepared with ultrapure water from a Millipore Milli-Q water purification system (18.2 MΩ cm resistivity).

The following commercially available antibodies were used: anti-BAX (Servicebio, cat: GB11007-1, 1:50 dilution, Wuhan, China), anti-Ki67 (Abcam, cat: Ab16667, 1:200 dilution, Cambridge, MA, USA), anti-VEGF (Servicebio, cat: GB11007-1, 1:100 dilution, Wuhan, China).
The following commercially available reagents were also used: SDS sample buffer (Sinopharm Group Chemical Reagent Co., Ltd., Beijing, China, cat: 30166428), 30% acrylamide (Biosharp, cat: BL513b, Hefei, China), Trise-Base (Biofavor biotech, cat: 1115GR500, Wuhan, China), Glycine (Biofavor biotech, cat: 1275GR500, Wuhan, China), methyl alcohol, NaCl, KCl, Na₂HPO₄.12H₂O, KH₂PO₄, acetic acid, Tween-20 (Sinopharm Group Chemical Reagent Co., Ltd., Beijing, China), protein markers (Thermo Fisher Scientific, Dover, DE, USA).

OTA standard (1 mg OTA solid powder) was purchased from Pribolab (Singapore), OchraTest^TM IACs were bought from Huaan Magnech Bio-Tech Co., Ltd. (Beijing, China). HPLC-grade methanol and acetonitrile were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Tween-20, Na₂HPO₄, and KCl were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). NaH₂PO₄ and KH₂PO₄ were purchased from Xilong Chemical Industry Co., Ltd. (Beijing, China). Other reagents and chemicals were of analytical grade and acquired from Beijing Chemical Works (Beijing, China); Wahaha purified water (Wahaha Group Co., Ltd, Hangzhou, China) was used.
PBS solution was prepared by dissolving 4 g NaCl, 0.1g KCl, 45 g Na₂HPO₄, and 0.1 g KH₂PO₄ in 500 mL of water with the pH being adjusted to 7.4 with 0.1 M HCL. PBT solution was prepared according to the following procedure; 8.5 mL of 0.2 M NaH₂PO₄ solution and 91.5 mL of 0.2 M Na₂HPO₄ solution were mixed into for preparing 0.2 M phosphate-buffer (PB) solution. Then, 25 mL of 0.2 M PB was added into 475 mL of water to get 0.01 M PB. Finally, 10 mL of Tween-20 was added into 500 mL of 0.01 M PB to yield PBT solution (pH 7.8).

The HM3030 XYZ three-dimensional film-spraying instrument, ZQ2002 microcomputer automatic chopping machine, and CTS350 CNC cutting machine were purchased from Shanghai Kinbio Tech. Co., Ltd. (Shanghai, China). The H3-16KR tabletop high-speed refrigerated centrifuge was purchased from Hunan Kecheng Instrument Equipment Co., Ltd. (Changsha, China), The MZC-2070M1 microwave oven was purchased from Haier Co., Ltd. (Qingdao, China). The HWS-24 Electric Thermostat Water Bath was purchased from Shanghai Huyan Industrial Co., Ltd. The refrigerator was purchased from Changhong Meiling Co., Ltd. (Hefei, China).
Trisodium phosphate, deoxyadenosine triphosphate (dATP), chloroauric acid (HAuCl₄⋅4H₂O), octylphenylpolyethylene glycol (Triton X-100), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS), Tween 20, trisodium citrate, sucrose, bovine serum albumin (BSA), n-butanol, and poly(ethylene glycol) (PEG) 4000 were procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); streptavidin (SA) was ordered from BBI Company. All oligonucleotide sequences are listed in the Supplementary Materials (Table S2). SX42 absorbent paper, CB06 glass-fiber paper, and an SMNF31-25 PVC bottom plate were bought from Shanghai Jinbiao Biological Co., Ltd. (Shanghai, China). CN140 nitrocellulose membrane was purchased from Sartorius Co., Ltd. (Goettingen, Germany).